Abstract: The aim of this proposal is to study and exploit the non-classical cross-linkages present in the peptidoglycan layer of bacteria. This layer is vital to the survival of bacilli and is maintained by linking peptide and glycan chains. One the most successful class of antibiotics in human clinical use, namely the [unreadable]-lactams and cephalosporins, targets the classical 4?3 linkages. The existing paradigm describes the peptidoglycan layer as a structure largely maintained by classical 4?3 linkages. Our recent studies of one of the most significant human pathogens, Mycobacterium tuberculosis (Mtb), has revealed that its peptidoglycan layer is composed of 3?3 (non-classical cross-links) and undergoes remodeling when the bacterium transitions from one growth phase to another. Other laboratories have recently identified this enzyme in bacteria such as E. coli and B. subtilis suggesting that this enzyme is widely present and vital for viability of bacteria. Emerging evidence from recent publications link remodeling of the peptidoglycan to contain 3?3 transpeptide bonds to adaptation during chronic phase of infection. In a short period of 2 years we have characterized a 3?3 transpeptidase and have now solved the molecular structure of the crystal containing native substrate. This proposal aims to develop and identify small molecules that mimic the substrate inhibit this novel transpeptidase activity. The inhibitor would be a promising new class of antibacterial drug Public Health Relevance: This proposal aims to investigate novel structures and molecules that are present in all important human bacterial pathogens. It is expected that findings from the proposed studies will lead to development of new interventions to kill bacteria. These drugs/anti-bacterials may be effective against a wide range of bacteria including those that are resistant to existing drugs.